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Abstract

Background

Subepithelial fibrosis is one of the most critical structural changes affecting bronchial
airway function during asthma. Eosinophils have been shown to contribute to the production
of pro-fibrotic cytokines, TGF-β and IL-11, however, the mechanism regulating this
process is not fully understood.

Objective

In this report, we investigated whether cytokines associated with inflammation during
asthma may induce eosinophils to produce pro-fibrotic cytokines.

Methods

Eosinophils were isolated from peripheral blood of 10 asthmatics and 10 normal control
subjects. Eosinophils were stimulated with Th1, Th2 and Th17 cytokines and the production
of TGF-β and IL-11 was determined using real time PCR and ELISA assays.

Results

The basal expression levels of eosinophil derived TGF-β and IL-11 cytokines were comparable
between asthmatic and healthy individuals. Stimulating eosinophils with Th1 and Th2
cytokines did not induce expression of pro-fibrotic cytokines. However, stimulating
eosinophils with Th17 cytokines resulted in the enhancement of TGF-β and IL-11 expression
in asthmatic but not healthy individuals. This effect of IL-17 on eosinophils was
dependent on p38 MAPK activation as inhibiting the phosphorylation of p38 MAPK, but
not other kinases, inhibited IL-17 induced pro-fibrotic cytokine release.

Conclusions

Th17 cytokines might contribute to airway fibrosis during asthma by enhancing production
of eosinophil derived pro-fibrotic cytokines. Preventing the release of pro-fibrotic
cytokines by blocking the effect of Th17 cytokines on eosinophils may prove to be
beneficial in controlling fibrosis for disorders with IL-17 driven inflammation such
as allergic and autoimmune diseases.

Keywords:

Introduction

Asthma is a chronic inflammatory disorder of the lung that is usually associated with
airway tissue remodelling. This term refers to the structural changes affecting lung
tissue which normally include epithelial detachment, increased airway smooth muscle
(ASM) mass, subepithelial fibrosis, mucous gland and goblet cell hyperplasia, vascular
changes, and edema
[1-4]. Subepithelial fibrosis is one of the most critical structural changes associated
with airway remodeling. In normal subjects, a loose array of collagen fibrils resides
beneath the basal membrane. In asthmatics, however, this layer is replaced by a dense
network of extra-cellular matrix (ECM) proteins including collagens
[5]. ECM protein deposition is known to be regulated by a number of cytokines and growth
factors including TGF-β
[6]. Several reports have shown that the majority of TGF-β1 mRNA positive cells in bronchial
biopsies of severe asthmatics were eosinophils
[7-9]. Eosinophils were also shown to produce IL-11 mRNA and protein
[10]. These reports suggested that eosinophils could play an important role in regulating
tissue fibrosis. IL-5 deficient mice experiments
[11] and human studies
[12] supported this hypothesis. In addition to lowering eosinophil levels, using anti-IL-5
antibodies was shown to be associated with reduced expression of ECM proteins particularly
tenascin, lumican, and procollagen III
[12].

Since its recent discovery, IL-17 has been described to be involved in various aspects
of asthma pathogenesis. Elevated IL-17A levels were shown to correlate with increased
airway hyper-responsiveness (AHR) in asthmatics
[13]. In fact, IL-17 was shown to modulate airway structural cells leading to tissue remodeling.
Over-expression of IL-17 F resulted in goblet cell hyperplasia and mucin gene expression
[14]. In addition, using an in vitro cell migration assay, Change et al. have recently
shown that Th17-associated cytokines IL-17A, IL-17 F, and IL-22 promote migration
of human ASMCs. These effects were shown to be mediated by selective activation of
receptors on ASMCs, with IL-17A and IL-17 F acting through p38 MAPK activation while
IL-22 acting through a distinct nuclear factor kB (NF-kB)–dependent signaling pathway
[15]. These studies indicated for a role of IL-17 in airway remodeling and hence in regulating
asthma pathogenesis.

Eosinophils have receptors for a number of mediators that are associated with asthma
including Th1, Th2, and Th17 cytokines
[16-18]. The expression of IL-17 cytokines was also associated with subepithelial fibrosis
[19-21]. In fact, Th17 cytokines were shown to trigger the expression of pro-fibrotic cytokines
in bronchial fibroblasts
[22]. We, hence, hypothesized that IL-17 cytokines may induce eosinophils to produce pro-fibrotic
cytokines. In this paper, we stimulated eosinophils, isolated from normal and asthmatic
subjects, with Th17 cytokines as well as a group of Th1 and Th2 cytokines known to
be associated with asthma. Eosinophil production of TGF-β and IL-11 pro-fibrotic cytokines
was then investigated.

Materials and methods

Study subjects

Ten subjects with severe asthma (6 males and 4 females, mean age 33.3 ± 2.6) who met
the criteria defined by ATS on refractory asthma
[23] were recruited. To be classified as severe asthmatics, patients must have had high-dose
inhaled corticosteroid: Budesonide 160 μg/twice a day (or equivalent) or daily anti-leukotriene
for >50% of the last year, and at least 1 other add-on therapy on daily basis for
the previous 12 months. They were also required to have two of the following criteria:
daily short-acting β-agonist, persistent FEV1 <60% and FEV1/FVC <75% predicted, 1
urgent visit or at least 3 steroid bursts in the previous year, prompt deterioration
with <25% steroid dose reduction, or previous near-fatal asthma within the last 3 years.
Subject characteristics are summarized in Table
1. Exclusion criteria included smoking history or any other pulmonary diseases or co-existing
medical conditions such as cardiac and renal diseases and uncontrolled hypertension.
Ten normal control subjects (6 males and 4 females, mean age: 38.2 ± 3.4) were also
recruited. All normal control subjects were non-smokers with normal lung function,
no history or symptoms of allergy and respiratory diseases, and were not taking any
medications for the preceding four weeks. The Ethics Committee of the King Khalid
University Hospital in Riyadh reviewed and approved the study, and all subjects recruited
signed written informed consent for the drawing of peripheral venous blood for the
isolation of eosinophils.

Statistical analysis

Data are presented as mean ± SD. Expression of pro-fibrotic cytokines was evaluated
using ANOVA followed by Bonferroni-Dunn post hoc test. Non-parametric Mann–Whitney U test (Systat, version 7.0, SPSS, Chicago, IL) was used to evaluate significance in
differential phosphorylation of MAPK. Values of p < 0.05 were considered statistically
significant.

Results

Th1 and Th2 cytokines do not induce expression of eosinophil derived pro-fibrotic
cytokines

The patho-physiological characteristics of lung tissue inflammation during severe
asthma differ significantly from those of the milder disease. While the airway tissues
of mild asthmatics usually present preferential Th2 cytokine profile
[28], those from severe asthmatics show a Th17 lymphocyte infiltration and elevated cytokine
levels, particularly Th1 cytokines (IFN-γ, IL-2), IL-17 and TGF-β
[29-31]. Many T-helper cytokines were shown to play a significant role in regulating TGF-β
expression and function in different types of cells
[32-34]. However, their direct role in regulating eosinophil ability to produce pro-fibrotic
cytokines was not studied. To investigate that, we first determined the basal expression
levels of pro-fibrotic cytokines within peripheral blood eosinophils of 10 asthmatic
and non-asthmatic individuals using real time RT-PCR. The levels of expression of
TGF-β and IL-11 mRNA in eosinophils isolated from asthmatic individuals (ct values:
TGF-β: 27.53 ± 0.21 IL-11: 28.80 ± 1.2) were comparable to those isolated from healthy
controls (ct values: TGF-β: 27.70 ± 0.29 IL-11: 29.56 ± 0.86) (Figure
1A). Eosinophil supernatant IL-11 and TGF-β cytokines levels were also determined in
the two groups using ELISA assay (Figure
1B). Similarly, no change in the secreted levels of these pro-fibrotic cytokines was
detected between the two groups. We then investigated whether Th1 and Th2 cytokines
play a role in regulating eosinophils pro-fibrotic cytokines production. To do that,
we stimulated 2×106 eosinophil cells isolated from 10 asthmatic as well as healthy individuals with Th1
(IL-2, IL-12, and IFN-γ), and Th2 (IL-4, IL-5, IL-9, and IL-13) cytokines as well
as GM-CSF for 4 hrs. Total RNA was then extracted from stimulated eosinophils and
the level of IL-11 and TGF-β was determined using real time RT-PCR. As shown in Figure
1C-D, stimulating asthmatic eosinophils with Th1 or Th2 cytokines did not affect TGF-β
(ct values range: 27.63 ± 0.21 to 27.58 ± 0.79) (Figure
1C) or IL-11 (ct values range: 28.67 ± 0.84 to 28.76 ± 0.18) (Figure
1D) m-RNA levels. Similar results were obtained at higher concentrations of Th1 and
Th2 cytokines (100 ng/ml) as well as for eosinophils isolated from healthy controls
(data not shown). These results indicated that neither Th1 nor Th2 cytokines play
a significant role in regulating expression of eosinophil derived pro-fibrotic cytokines.

Figure 1.Basal expression of pro-fibrotic cytokines by human eosinophils isolated from asthmatic
and controls subjects. Eosinophils were isolated from 10 asthmatic and 10 controls subjects and total RNA
was extracted from 2×106 million cells and quantified using real-time PCR. A: Level of expression of TGF-β1 and IL-11 mRNA in eosinophils of asthmatic versus
control subjects (n = 10). B: Levels of TGF-β1 and IL-11 cytokines within the supernatant of un-stimulated eosinophils
(n = 10) as determined by ELISA assay. (C-D) Effect of Th1 and Th2 cytokines on asthmatic eosinophil TGF-β1 and IL-11 transcripts
levels. Level of expression of TGF-β1 (C) and IL-11 (D) mRNA as quantified by real-time PCR following 4 hours exposure to mediators. Data
is presented as percentage of basal expression (n = 10).

Figure 2.IL-17 and IL-23 enhance eosinophil expression of pro-fibrotic cytokines. (A) Surface expression of IL-17R on eosinophils (1×106 cells) isolated from healthy and asthmatics was determined by flow cytometry. Blots
are representative data for eosinophils isolated from one healthy control and one
asthmatic patient. The graph shows arithmetic mean ± SD of IL-17R positive eosinophils
as percentage of total eosinophils (n = 5). 2×106 peripheral blood eosinophils isolated from 10 asthmatic and 10 controls subjects
were stimulated with IL-17A, F, and IL-23 (50 ng/ml or 25 ng/ml) alone or in combination
for 4 hrs. Total RNA was extracted and mRNA levels of TGF-β and IL-11 were then quantified
using real-time PCR. mRNA expression levels of TGF-β (B) and IL-11 (C) were normalized with GAPDH for asthmatic versus healthy individuals.

P38 mitogen-activated protein kinase (MAPK), being at a critical junction of the IL-17
signaling pathways, has been shown by various reports to be a key regulator element
for the activity of IL-17 cytokines
[15,16,36]. To study the mechanism behind Th17 cytokines enhancement of eosinophil derived TGF-β
production, eosinophils were isolated from peripheral blood of 10 asthmatic patients
as described above. 2×106 cells were treated, or not, with p38 MAPK or PI3K inhibitors (SB2035802 and PI103,
respectively), or diluent control (DMSO) 2 hours prior to stimulation with IL-17.
As shown in Figure
4, inhibiting phosphorylation of p38 MAPK significantly decreased the level of TGF-β
(P = 0.011 (IL-17A + F); P = 0.015 (IL-17A + F + 23); n = 10) and IL-11 (P = 0.021
(IL-17A + F); P = 0.026 (IL-17A + F + 23); n = 10) secreted into eosinophil supernatants
24 hrs following Th17 cytokine stimulation (Figure
4A, B). This blocking effect was only specific to p38 MAPK as diluent control or inhibitor
of another kinase (PI3K) did not affect the supernatant levels of TGF-β and IL-11
(P = NS; n = 10). This data indicated that p38 MAPK activation is critical for IL-17
induced eosinophil derived pro-fibrotic cytokine production. To confirm p38 MAPK phosphorylation
following treatment with IL-17 cytokines, 2×106 eosinophil cell were treated with IL-17A + F (50 ng/ml each) for 0, 10 and 20 minutes
and the level of p38 MAPK phosphorylation was then determined using western analysis.
As shown in Figure
4C, stimulating eosinophils with a combination of IL-17A and IL-17 F resulted in phosphorylation
of p38 MAPK which seems to peak at 10 minutes (3.5 fold increase, p = 0.039). Inhibiting
p38 MAPK, PI3K, or ERK1/2, however, did not interfere with the ability of IL-23 to
stimulate eosinophil to produce pro-fibrotic cytokines. This indicated that IL-23
may use other mechanisms to stimulate pro-fibrotic cytokine release that need to be
further investigated.

Figure 4.P38 MAP Kinase activation is required for IL-17 enhancement of eosinophil derived
pro-fibrotic cytokines. Eosinophils were isolated from peripheral blood of 10 asthmatic patients and 2×106/ml cells were treated, or not, with p38 MAPK or PI3K inhibitors (SB2035802 and PI103,
respectively) 2 hours prior to stimulation with IL-17 (50 ng/ml). Levels of TGF-β
(A) and IL-11 (B) in the supernatant of stimulated eosinophils were then determined 24 hrs following
Th17 cytokine stimulation using ELISA assay (n = 10). (C) Induction of p38 MAPK phosphorylation by a combination of IL-17A and IL-17 F (50 ng/ml
each) is detected by western analysis. The western data shown represent one of similar
results from 4 independent experiments. * = p < 0.05 compared to non-stimulated (NS).
** = p < 0.05 compared to stimulated not inhibited.

Discussion

Eosinophils constitute a major source of TGF-β in asthmatic lung tissue
[7-9]. Reduction of lung eosinophilia by anti–IL-5 therapy in humans
[12] or genetic knock down in mice
[11] significantly reduced airway fibrosis and pulmonary TGF-β1 levels. Here, we show,
for the first time, that Th17 cytokines enhance eosinophil derived TGF-β and IL-11
production. This effect of Th17 cytokines was prominent on eosinophils isolated from
asthmatics but not healthy subjects. Our results clearly demonstrate that eosinophils
constitute an additional site of action for Th17 cytokines in asthma supporting a
role for IL-17 in regulating fibrosis and airway remodeling.

Although Th2 (IL-4, IL-5, and IL-13) cytokines has earlier been reported to regulate
the expression of TGF-β1 by eosinophils
[37,38], other studies had shown no effect of these cytokines on TGF-β expression
[39]. Our results support the latest reports as we did not see any increase in TGF-β or
IL-11 mRNA or protein expression following stimulation with Th2 cytokines. Similarly,
Th1 cytokines had no effect on eosinophil derived TGF-β expression. In fact, IFN-γ
was previously shown to inhibit TGF-β production in human airway epithelial cells
which is in consistence with our findings
[38].

The enhancement of eosinophil derived pro-fibrotic cytokine release upon IL-17 cytokines
stimulation was only significant in eosinophils isolated from asthmatic individuals.
Although there was a slight upregulation of TGF-β and IL-11 expression in eosinophils
isolated from healthy individuals upon IL-17 stimulation, this increase did not reach
significance. Peripheral blood eosinophils of asthmatic patients were shown to be
primed compared to those of healthy subjects
[40-42] which may render them more susceptible to IL-17 effect. Our results suggest that
IL-17 cytokines enhance pro-fibrotic activity of activated, such as in the case of
allergic and auto-immune diseases, but not resting eosinophils. Furthermore, our data
indicated that asthmatic eosinophils may express higher levels of IL-17R than those
of healthy controls (Figure
2A). IL-23 was shown to increase expression of IL-17RA and IL-17RC in eosinophils
[16] and hence this observed potential increase in IL-17R in asthmatic eosinophils could
be due to increased serum IL-23 in those patients. Serum levels of IL-23 were shown
to inversely correlate with level of pulmonary function (FEV1) of asthmatic patients
in various reports
[43,44]. This may indicate that, due to the expected increase in serum IL-23 with asthma
severity, eosinophils isolated from mild and moderate asthmatic patients may express
higher levels of IL-17 receptors than eosinophils of healthy controls but lower than
those of severe asthmatic patients. Understanding the correlation between asthmatic
patients’ IL-23 serum levels, the expression of IL-17R on peripheral blood eosinophils,
and the severity of asthma requires further investigations.

Eosinophils are known to produce IL-17 cytokines
[22] and IL-23 was shown to stimulate the expression of IL-17A cytokine
[45]. This may indicate that IL-23 could stimulate eosinophils release of pro-fibrotic
cytokines indirectly by triggering their release of IL-17A. This possibility, however,
needs to be further investigated.

Stimulating eosinophils with IL-17 cytokines at a physiologically relevant concentration
(25 ng/ml) resulted in an increase in TGF-β and IL-11 production although not to a
significant levels (Figure
3). While stimulating eosinophils with either IL-17A or F alone did not enhance a significant
increase in pro-fibrotic cytokines, using a combination of both cytokines did indicating
an additive effect. Since both IL-17A and IL-17 F share the same IL-17R receptor
[46], a concentration of around 25 ng/ml or more of each IL-17 cytokine seems to be required
for efficient eosinophil derived pro-fibrotic cytokine release. This is more likely
to be achieved in vivo through the additive effect of IL-17A and F rather than a high
concentration of a single IL-17 cytokine alone.

Accumulating evidences from various reports indicate for a key role of p38 MAPK pathway
in IL-17 cytokine activity on structural and inflammatory cells in asthma
[15,36]. Binding of IL-17A and F to the IL-17RA and RC receptors on target cells triggers
the recruitment of the U-box E3 ubiquitin ligase Act1 (CIKS). Act1 will in turn recruit
TGF-β activated kinase that serves as the template for the activation of the transcription
factors NF-kB, CEBPb (beta), as well as the MAPK pathways ERK1/ERK2 and p38 MAPK
[47]. P38 MAPK, ERK, and JNK pathways were shown to regulate TGF-β transcription each
in response to different stimuli
[48]. Our data suggest that IL-17 cytokines stimulate TGF-β transcription via the activation
of p38 MAPK but not PI3K or ERK1/2 MAPK (data not shown) pathways. IL-23, however,
seems to use another mechanism as inhibiting those pathways did not affect its ability
to stimulate TGF-β and IL-11 production.

Conclusions

Data presented herein suggest a new role for Th17 cytokines in airway remodeling during
asthma. IL-17 cytokines seem to contribute to airway tissue fibrosis by enhancing
production of eosinophil derived pro-fibrotic cytokines. This role of IL-17 was dependent
on p38 MAPK activation. Therefore, upstream activators of p38 MAPK within the IL-17R
pathway may represent an attractive target in corticosteroid-unresponsive diseases
[49,50]. Preventing the release of TGF-β by blocking the effect of IL-17 on eosinophils may
also prove efficient in controlling fibrosis for disorders with IL-17 driven inflammation
such as allergic and autoimmune diseases.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

SM carried out the real-time PCR for Th17 experiments and participated in the design
of the study. SL carried out the real-time PCR and ELISA for Th1 and Th2 experiments.
AVT participated in the design of the study and performed the statistical analysis.
MAP carried out the ELISA for Th17 experiments. HJ contributed in recruiting patients
to the study. QH contributed in study design and data analysis. RH conceived of the
study, lead efforts on its design and coordination and finalized the manuscript draft.
All authors read and approved the final manuscript.

Acknowledgements

This study was supported by a grant from the National Plan for Sciences and Technology,
King Saud University, Riyadh, Saudi Arabia (grant number 09-BIO907-02). The funders
had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.